The invention relates to a novel recombinant S-adenosyl homocysteinase (SAHH) and methods of using such SAHH. The invention is also directed to diagnostic methods to monitor subjects who have been administered S-adenosylmethionine (SAM), using SAHH. The improved methods of the invention provide rapid and accurate assessment of the concentrations of SAM.
The administration of S-adenosylmethionine (SAM) as a xe2x80x9cnutraceuticalxe2x80x9d or as a prescribed medication has recently been suggested as an antidepressant, a preventative or therapeutic component in ameliorating liver disease, and a means to diminish the symptoms of arthritis. The mechanism whereby SAM is believed to act is not understood completely, but it is believed that the relative concentrations of SAM and homocysteine, which is a metabolic product of SAM, affect methylation levels which, in turn, have profound physiological effects. In view of the importance of this drug, it would be desirable to have a reliable and easily performed method to monitor the concentration of the administered pharmaceutical. The present invention provides an improved method to assess therapeutic levels of SAM in subjects administered this drug using S-adenosyl homocysteinase (SAHH). The present invention is also directed to a recombinantly produced SAHH that differs from the previously reported SAHH.
S-adenosylhomocysteinase (S-adenosylhomocysteine hydrolase; SAHH, EC 3.3.1.1) catalyses the reversible conversion of SAH to homocysteine and adenosine (de la Haba and Cantoni, 1959). Various structural analogues of adenosine inactivate SAHH from a number of organisms, resulting in cytotoxicity (Ueland, 1982). Inhibition of SAHH activity by the nucleoside analogues depends on the inhibitor structure as well as the source of the enzyme. SAHH was initially cloned from the Trichomonas vaginalis gene and previously characterized (Bagnara et al., 1996).
Minotto, L., Ko, G. -A., Edwards, M. R., and Bagnara, A. S. [Trichomonas vaginalis Expression and characterization of recombinant S-adenosylhomocysteinase. Experimental Parasitology 90, 175-180, 1998] have further characterized the T. vaginalis SAHH. The gene encoding S-adenosylhomocysteinase in Trichomonas vaginalis was expressed on pQE-30 in Escherichia coli to facilitate the characterization of the enzyme.
A 6xc3x97 His N-terminal tag expression system (QIAGEN) enabled one-step purification of six mg of rSAHH, obtained from a 100-ml bacterial culture by affinity chromatography using a nickel-NTA matrix. The recombinant enzyme was found to have molecular weight of approximately 56,000. Properties of rSAHH include a similar apparent Km for adenosine of 20-25 xcexcM for the recombinant and similar inhibition/inactivation patterns adenosine analogues such as arabinosyl adenine (ara-A).
The results of Minotto et al., 1998, differ from the finding of others who have shown that the hydrolase can exist in various oligomeric forms depending on the source of the enzyme. The SAHH activity from prokaryotes is as a hexamer (Shimizu et al., 1984) or tetramer (Porcelli et al., Biochim. et Biophys. Acta, 1164, 179-188, 1993). The enzyme from rat liver (Fujioka and Takata, J Biol. Chem., 256, 1631-1635, 1981), calf liver (Richards et al., J. Biol. Chem., 253, 4476-4480, 1978), and other animal sources (Doskeland and Ueland, Biochem et Biophys Acta 708, 185-193, 1982) is tetrameric but with uncertainty whether the subunits are identical or similar. SAHH from a plant source is functional as a homodimer (Guranowski and Pawelkiewicz, Eur. J. Biochem. 80, 517-523, 1977). This is the first report of an SAHH activity being functional in the monomeric form (Minotto et al., 1998).
The present invention relates to an improved and novel method for analyzing SAM levels in a sample. In one aspect of the invention, this method may be used to assay therapeutic levels of SAM in a sample from a subject such as, but not limited to, a patient being administered this compound. The method may also be used, to assay SAM levels in a biological fluid such as, but not limited to, blood or other biological fluids of a subject. Such methods may be conducted in vivo, such as in the bloodstream, or in vitro, such as with a sample taken from a subject. The methods may be used as part of a diagnostic protocol or as part of a therapeutic protocol. As part of a therapeutic protocol, the methods may serve in part to monitor the conditions or progress of the therapy.
In one embodiment of the invention, the assay method may be performed by contacting a sample with glycine N-methyltransferase (GMT), glycine, and SAHH activity. Determination of SAM levels in the sample may then be made by measuring one or more reaction products in the sample, wherein the amount of reaction product(s) is directly proportional to SAM levels in the sample. In one embodiment of the invention, the reaction product homocysteine (HC) is measured directly or indirectly. Indirect measurements of HC may be made by any means including, but not limited to, treatment with homocysteinase (HCYase) and measuring the levels of one or more reaction products (e.g. alpha keto glutarate, H2S, or NH3). The H2S reaction product may be measured directly or indirectly by measuring absorbance or fluorescence. One means of measuring fluorescence is by use of a fluorescence generating reagent.
The invention also provides a novel SAHH, nucleic acids that encode it, compositions comprising it, and methods for its preparation and use. The SAHH contains an amino acid sequence encoded by SEQ ID NO:1. Nucleic acids which encode the SAHH of the invention may be placed in any appropriate nucleic acid vector for propagation, amplification or expression. The nucleic acids may also be operably linked to other nucleic acids to permit the expression of the SAHH covalently linked to one or more additional amino acids. The additional amino acids result in the production of a hybrid or chimeric protein comprising SAHH. In one preferred embodiment of the invention, the additional amino acids are those of a histidine tag (His tag) which improves subsequent purification of the SAHH of the invention.
The nucleic acids of the invention may be introduced into any appropriate host cell or organism, such as, but not limited to, bacteria, fungi, and higher eukaryotic cells. These cells may be used to recombinantly express the nucleic acids of the invention, optionally followed by isolation and/or purification of the expressed protein. Alternatively, the nucleic acids may also be expressed by use of in vitro expression systems.
Purification of the SAHH of the invention may be by any convenient or appropriate means such as, but not limited to, precipitation and/or chromatography. In a preferred embodiment of the invention, the purification is performed in whole or in part by affinity chromatography based on interaction with a His tag. In another preferred embodiment of the invention, the SAHH is purified such that it appears as a single band when analyzed by SDS polyacrylamide gel electrophoresis.
The SAHH of the invention may also be formulated into compositions, such as those comprising pharmaceutical agents or excipients. The SAHH may also be used in the methods of the invention, such as the assay methods described above, as well as additional methods such as that for assaying homocysteine to SAH conversion in a sample to measure homocysteine levels. In another aspect of the invention, the SAHH may be used in methods of depleting excess homocysteine in a sample in vivo or in vitro by conversion to SAH. Of course the samples of the invention may be any biological fluid of interest.